Upon exposure to genotoxic stress, cells activate DNA damage response (DDR) that coordinates DNA repair with a temporal arrest in the cell cycle progression. DDR is triggered by activation of ataxia telangiectasia mutated/ataxia telangiectasia and Rad3-related protein kinases that phosphorylate multiple targets including tumor suppressor protein tumor suppressor p53 (p53). In addition, DNA damage can activate parallel stress response pathways [such as mitogen-activated protein kinase p38 alpha (p38)/MAPK-activated protein kinase 2 (MK2) kinases] contributing to establishing the cell cycle arrest. Wild-type p53-induced phosphatase 1 (WIP1) controls timely inactivation of DDR and is needed for recovery from the G2 checkpoint by counteracting the function of p53. Here, we developed a simple in vitro assay for testing WIP1 substrates in nuclear extracts. Whereas we did not detect any activity of WIP1 toward p38/MK2, we confirmed p53 as a substrate of WIP1. Inhibition or inactivation of WIP1 in U2OS cells increased phosphorylation of p53 at S15 and potentiated its acetylation at K382. Further, we identified Deleted in breast cancer gene 1 (DBC1) as a new substrate of WIP1 but surprisingly, depletion of DBC1 did not interfere with the ability of WIP1 to regulate p53 acetylation. Instead, we have found that WIP1 activity suppresses p53-K382 acetylation by inhibiting the interaction between p53 and the acetyltransferase p300. Newly established phosphatase assay allows an easy comparison of WIP1 ability to dephosphorylate various proteins and thus contributes to identification of its physiological substrates.

Cancer Cell Biology Institute of Molecular Genetics of the Czech Academy of Sciences Prague Czech Republic

Division of Cell Biology Netherlands Cancer Institute Amsterdam The Netherlands

Faculty of Science Charles University Prague Czech Republic

Zobrazit více v PubMed

Jackson SP & Bartek J (2009) The DNA-damage response in human biology and disease. Nature 461, 1071-1078.

Bartek J & Lukas J (2003) Chk1 and Chk2 kinases in checkpoint control and cancer. Cancer Cell 3, 421-429.

Blackford AN & Jackson SP (2017) ATM, ATR, and DNA-PK: the trinity at the heart of the DNA damage response. Mol Cell 66, 801-817.

Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER III, Hurov KE, Luo J, Bakalarski CE, Zhao Z, Solimini N, Lerenthal Y et al. (2007) ATM and ATR substrate analysis reveals extensive protein networks responsive to DNA damage. Science 316, 1160-1166.

Bensimon A, Schmidt A, Ziv Y, Elkon R, Wang SY, Chen DJ, Aebersold R & Shiloh Y (2010) ATM-dependent and -independent dynamics of the nuclear phosphoproteome after DNA damage. Sci Signal 3, rs3.

Choi BK, Fujiwara K, Dayaram T, Darlington Y, Dickerson J, Goodell MA & Donehower LA (2020) WIP1 dephosphorylation of p27(Kip1) Serine 140 destabilizes p27(Kip1) and reverses anti-proliferative effects of ATM phosphorylation. Cell Cycle 19, 479-491.

el-Deiry WS, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, Lin D, Mercer WE, Kinzler KW & Vogelstein B (1993) WAF1, a potential mediator of p53 tumor suppression. Cell 75, 817-825.

Reinhardt HC, Aslanian AS, Lees JA & Yaffe MB (2007) p53-deficient cells rely on ATM- and ATR-mediated checkpoint signaling through the p38MAPK/MK2 pathway for survival after DNA damage. Cancer Cell 11, 175-189.

Manke IA, Nguyen A, Lim D, Stewart MQ, Elia AE & Yaffe MB (2005) MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. Mol Cell 17, 37-48.

Bulavin D, Higashimoto Y, Popoff I, Gaarde W, Basrur V & Potapova O (2001) Initiation of a G2/M checkpoint after ultraviolet radiation requires p38 kinase. Nature 411, 102-107.

Shaltiel IA, Aprelia M, Saurin AT, Chowdhury D, Kops GJPL, Voest EE & Medema RH (2014) Distinct phosphatases antagonize the p53 response in different phases of the cell cycle. Proc Natl Acad Sci USA 111, 7313-7318.

Fiscella M, Zhang H, Fan S, Sakaguchi K, Shen S, Mercer WE, Vande Woude GF, O'Connor PM & Appella E (1997) Wip1, a novel human protein phosphatase that is induced in response to ionizing radiation in a p53-dependent manner. Proc Natl Acad Sci USA 94, 6048-6053.

Shreeram S, Demidov ON, Hee WK, Yamaguchi H, Onishi N, Kek C, Timofeev ON, Dudgeon C, Fornace AJ, Anderson CW et al. (2006) Wip1 phosphatase modulates ATM-dependent signaling pathways. Mol Cell 23, 757-764.

Macurek L, Lindqvist A, Voets O, Kool J, Vos H & Medema R (2010) Wip1 phosphatase is associated with chromatin and dephosphorylates gammaH2AX to promote checkpoint inhibition. Oncogene 29, 2281-2291.

Burdova K, Storchova R, Palek M & Macurek L (2019) WIP1 promotes homologous recombination and modulates sensitivity to PARP inhibitors. Cells 8, 1258.

Jaiswal H, Benada J, Müllers E, Akopyan K, Burdova K, Koolmeister T, Helleday T, Medema RH, Macurek L & Lindqvist A (2017) ATM/Wip1 activities at chromatin control Plk1 re-activation to determine G2 checkpoint duration. EMBO J 36, 2161-2176.

Fujimoto H, Onishi N, Kato N, Takekawa M, Xu X & Kosugi A (2006) Regulation of the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase. Cell Death Differ 13, 1170-1180.

Lu X, Nannenga B & Donehower L (2005) PPM1D dephosphorylates Chk1 and p53 and abrogates cell cycle checkpoints. Genes Dev 19, 1162-1174.

Lindqvist A, de Bruijn M, Macurek L, Bras A, Mensinga A & Bruinsma W (2009) Wip1 confers G2 checkpoint recovery competence by counteracting p53-dependent transcriptional repression. EMBO J 28, 3196-3206.

Krenning L, Feringa FM, Shaltiel IA, van den Berg J & Medema RH (2014) Transient activation of p53 in G2 phase is sufficient to induce senescence. Mol Cell 55, 59-72.

Pechackova S, Burdova K, Benada J, Kleiblova P, Jenikova G & Macurek L (2016) Inhibition of WIP1 phosphatase sensitizes breast cancer cells to genotoxic stress and to MDM2 antagonist nutlin-3. Oncotarget 7, 14458-14475.

Emelyanov A & Bulavin DV (2015) Wip1 phosphatase in breast cancer. Oncogene 34, 4429-4438.

Bulavin DV, Demidov ON, Saito SI, Kauraniemi P, Phillips C, Amundson SA, Ambrosino C, Sauter G, Nebreda AR, Anderson CW et al.(2002) Amplification of PPM1D in human tumors abrogates p53 tumor-suppressor activity. Nat Genet 31, 210-215.

Zhang L, Chen LH, Wan H, Yang R, Wang Z, Feng J, Yang S, Jones S, Wang S, Zhou W et al. (2014) Exome sequencing identifies somatic gain-of-function PPM1D mutations in brainstem gliomas. Nat Genet 46, 726-730.

Kleiblova P, Shaltiel IA, Benada J, Sevc\̌ík J, Pechác\̌ková S, Pohlreich P, Voest EE, Dundr P, Bartek J, Kleibl Z et al. (2013) Gain-of-function mutations of PPM1D/Wip1 impair the p53-dependent G1 checkpoint. J Cell Biol 201, 511-521.

Richter M, Dayaram T, Gilmartin AG, Ganji G, Pemmasani SK, Van Der Key H, Shohet JM, Donehower LA & Kumar R (2015) WIP1 phosphatase as a potential therapeutic target in neuroblastoma. PLoS One 10, e0115635.

Gilmartin AG, Faitg TH, Richter M, Groy A, Seefeld MA, Darcy MG, Peng X, Federowicz K, Yang J, Zhang S-Y et al. (2014) Allosteric Wip1 phosphatase inhibition through flap-subdomain interaction. Nat Chem Biol 10, 181-187.

Yamaguchi H, Minopoli G, Demidov ON, Chatterjee DK, Anderson CW, Durell SR & Appella E (2005) Substrate specificity of the human protein phosphatase 2Cdelta, Wip1. Biochemistry 44, 5285-5294.

Yamaguchi H, Durell SR, Chatterjee DK, Anderson CW & Appella E (2007) The Wip1 phosphatase PPM1D dephosphorylates SQ/TQ motifs in checkpoint substrates phosphorylated by PI3K-like kinases. Biochemistry 46, 12594-12603.

Kim S, Lim D, Canman C & Kastan M (1999) Substrate specificities and identification of putative substrates of ATM kinase family members. J Biol Chem 274, 37538-37543.

Kahn JD, Miller PG, Silver AJ, Sellar RS, Bhatt S, Gibson C, McConkey M, Adams D, Mar B, Mertins P et al. (2018) PPM1D truncating mutations confer resistance to chemotherapy and sensitivity to PPM1D inhibition in hematopoietic cells. Blood 132, 1095.

Takekawa M, Adachi M, Nakahata A, Nakayama I, Itoh F, Tsukuda H, Taya Y & Imai K (2000) p53-inducible wip1 phosphatase mediates a negative feedback regulation of p38 MAPK-p53 signaling in response to UV radiation. EMBO J 19, 6517-6526.

Brazina J, Svadlenka J, Macurek L, Andera L, Hodny Z, Bartek J & Hanzlikova H (2015) DNA damage-induced regulatory interplay between DAXX, p53, ATM kinase and Wip1 phosphatase. Cell Cycle 14, 375-387.

Moon S, Lin L, Zhang X, Nguyen T, Darlington Y, Waldman A, Lu X & Donehower LA (2010) Wildtype p53-induced phosphatase 1 dephosphorylates histone variant gamma-H2AX and suppresses DNA double strand break repair. J Biol Chem 285, 12935-12947.

Reinhardt HC, Hasskamp P, Schmedding I, Morandell S, van Vugt MA, Wang X, Linding R, Ong SE, Weaver D, Carr SA et al. (2010) DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization. Mol Cell 40, 34-49.

Söderberg O, Gullberg M, Jarvius M, Ridderstråle K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG et al. (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3, 995-1000.

Zannini L, Buscemi G, Kim JE, Fontanella E & Delia D (2012) DBC1 phosphorylation by ATM/ATR inhibits SIRT1 deacetylase in response to DNA damage. J Mol Cell Biol 4, 294-303.

Zhao W, Kruse JP, Tang Y, Jung SY, Qin J & Gu W (2008) Negative regulation of the deacetylase SIRT1 by DBC1. Nature 451, 587-590.

Kim JE, Chen J & Lou Z (2008) DBC1 is a negative regulator of SIRT1. Nature 451, 583-586.

Deng K, Liu L, Tan X, Zhang Z, Li J, Ou Y, Wang X, Yang S, Xiang R & Sun P (2020) WIP1 promotes cancer stem cell properties by inhibiting p38 MAPK in NSCLC. Signal Transduct Target Ther 5, 36.

Bulavin DV, Phillips C, Nannenga B, Timofeev O, Donehower LA, Anderson CW, Appella E & Fornace AJ (2004) Inactivation of the Wip1 phosphatase inhibits mammary tumorigenesis through p38 MAPK-mediated activation of the p16Ink4a-p19Arf pathway. Nat Genet 36, 343-350.

Demidov ON, Kek C, Shreeram S, Timofeev O, Fornace AJ, Appella E & Bulavin DV (2006) The role of the MKK6//p38 MAPK pathway in Wip1-dependent regulation of ErbB2-driven mammary gland tumorigenesis. Oncogene 26, 2502-2506.

Shreeram S, Demidov O, Hee W, Yamaguchi H, Onishi N & Kek C (2006) Wip1 phosphatase modulates ATM-dependent signaling pathways. Mol Cell 23, 757-764.

Lu X, Ma O, Nguyen T-A, Jones SN, Oren M & Donehower LA (2007) The Wip1 phosphatase acts as a gatekeeper in the p53-Mdm2 autoregulatory loop. Cancer Cell 12, 342-354.

Tang Y, Zhao W, Chen Y, Zhao Y & Gu W (2008) Acetylation is indispensable for p53 activation. Cell 133, 612-626.

Luo J, Li M, Tang Y, Laszkowska M, Roeder RG & Gu W (2004) Acetylation of p53 augments its site-specific DNA binding both in vitro and in vivo. Proc Natl Acad Sci USA 101, 2259-2264.

Barlev NA, Liu L, Chehab NH, Mansfield K, Harris KG, Halazonetis TD & Berger SL (2001) Acetylation of p53 activates transcription through recruitment of coactivators/histone acetyltransferases. Mol Cell 8, 1243-1254.

Gu W & Roeder RG (1997) Activation of p53 sequence-specific DNA binding by acetylation of the p53 C-terminal domain. Cell 90, 595-606.

Jenkins LM, Yamaguchi H, Hayashi R, Cherry S, Tropea JE, Miller M, Wlodawer A, Appella E & Mazur SJ (2009) Two distinct motifs within the p53 transactivation domain bind to the Taz2 domain of p300 and are differentially affected by phosphorylation. Biochemistry 48, 1244-1255.

Feng H, Jenkins LM, Durell SR, Hayashi R, Mazur SJ, Cherry S, Tropea JE, Miller M, Wlodawer A, Appella E et al. (2009) Structural basis for p300 Taz2-p53 TAD1 binding and modulation by phosphorylation. Structure (London, England: 1993) 17, 202-210.

Avantaggiati ML, Ogryzko V, Gardner K, Giordano A, Levine AS & Kelly K (1997) Recruitment of p300/CBP in p53-dependent signal pathways. Cell 89, 1175-1184.

Sakaguchi K, Herrera JE, Saito S, Miki T, Bustin M, Vassilev A, Anderson CW & Appella E (1998) DNA damage activates p53 through a phosphorylation-acetylation cascade. Genes Dev 12, 2831-2841.

Teufel DP, Bycroft M & Fersht AR (2009) Regulation by phosphorylation of the relative affinities of the N-terminal transactivation domains of p53 for p300 domains and Mdm2. Oncogene 28, 2112-2118.

Li Q, Hao Q, Cao W, Li J, Wu K, Elshimali Y, Zhu D, Chen Q-H, Chen G, Pollack JR et al. (2019) PP2Cδ inhibits p300-mediated p53 acetylation via ATM/BRCA1 pathway to impede DNA damage response in breast cancer. Sci Adv 5, eaaw8417.

Li J, Bonkowski MS, Moniot S, Zhang D, Hubbard BP, Ling AJ, Rajman LA, Qin B, Lou Z, Gorbunova V et al. (2017) A conserved NAD(+) binding pocket that regulates protein-protein interactions during aging. Science 355, 1312-1317.

Uematsu N, Weterings E, Yano K, Morotomi-Yano K, Jakob B, Taucher-Scholz G, Mari PO, van Gent DC, Chen BP & Chen DJ (2007) Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks. J Cell Biol 177, 219-229.

Crowe JL, Wang XS, Shao Z, Lee BJ, Estes VM & Zha S (2020) DNA-PKcs phosphorylation at the T2609 cluster alters the repair pathway choice during immunoglobulin class switch recombination. Proc Natl Acad Sci USA 117, 22953-22961.

Shao Z, Flynn RA, Crowe JL, Zhu Y, Liang J, Jiang W, Aryan F, Aoude P, Bertozzi CR, Estes VM et al. (2020) DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis. Nature 579, 291-296.

Jiang W, Estes VM, Wang XS, Shao Z, Lee BJ, Lin X, Crowe JL & Zha S (2019) Phosphorylation at S2053 in Murine (S2056 in Human). DNA-PKcs is dispensable for lymphocyte development and class switch recombination. J Immunol 203, 178-187.

von Morgen P, Burdova K, Flower TG, O'Reilly NJ, Boulton SJ, Smerdon SJ, Macurek L & Horejsi Z (2017) MRE11 stability is regulated by CK2-dependent interaction with R2TP complex. Oncogene 36, 4943-4950.

Masuda T, Tomita M & Ishihama Y (2008) Phase transfer surfactant-aided trypsin digestion for membrane proteome analysis. J Proteome Res 7, 731-740.

Cox J, Hein MY, Luber CA, Paron I, Nagaraj N & Mann M (2014) Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol Cell Proteomics 13, 2513-2526.

PPM1D activity promotes cellular transformation by preventing senescence and cell death

Phosphorylation of TRF2 promotes its interaction with TIN2 and regulates DNA damage response at telomeres